In the erythrocyte, methemoglobin (metHb) is reduced to ferroHb primarily by reaction with ferrocytochrome b5 which mediates the transfer of electrons from NADH-dependent metHb reductase. This recycling of the non-functional metHb is essential to prevent accumulation of the oxidized protein during the 120 day life span of the red cell since 0.5-3% of circulating Hb is converted to metHb each day. Deficiencies in this reductase activity result in methemoglobinemia. Combined erythrocyte and brain deficiencies (related enzymes occur in most tissues) produce methemoglobinemia, mental retardation and early death. As the 3-dimensional structures of cytochrome b5 and metHb are known to high resolution, we are developing a detailed model for the electron transfer reaction between these two proteins that should define an unexplored area of Hb physiology and provide a working model for electron transfer reactions between other metalloproteins (e.g., the reaction of cytochrome c with cytochrome oxidase). Parallel studies are also outlined for the simpler but closely related reaction between the well-characterized proteins cytochrome b5 and cytochrome c. Features of this reaction mechanism that will be investigated include (1) identification of amino acid residues on the two proteins that are responsible for protein-protein recognition through use of chemical modification and crosslinking techniques, (2) determination of stability of the cytochrome b5-metHb complex at physiological pH and ionic strength by fluorescence quenching titrations, (3) estimation of the distance between redox centers of the two proteins by energy transfer measurements and determination of the functional dependence of the rate of electron transfer on this distance, and (4) preparation and structural and functional characterization of crosslinked metHb-cytochrome b5 complexes. In addition, we will explore the possibility that the heme exchange reaction between metHb and apocytochrome b5 is a reasonable model for the transfer of heme from metHb to hemopexin, a heme-scavenging protein from plasma known to have surface properties and heme-binding characterization very similar to these of cytochrome b5.